Viruses from the genus Enterovirus (EV) of the Picornaviridae family are known to cause diseases such as hand foot and mouth disease (HFMD), respiratory diseases, encephalitis and myocarditis. The capsid of EV is an attractive target for the development of direct-acting small molecules that can interfere with viral entry. Some of the capsid binders have been evaluated in clinical trials but the majority have failed due to insufficient efficacy or unacceptable off-target effects. Furthermore, most of the capsid binders exhibited a low barrier to resistance. Alternatively, host-targeting inhibitors such as peptides derived from the capsid of EV that can recognize cellular receptors have been identified. However, the majority of these peptides displayed low anti-EV potency (µM range) as compared to the potency of small molecule compounds (nM range). Nonetheless, the development of anti-EV peptides is warranted as they may complement the small-molecules in a drug combination strategy to treat EVs. Lastly, structure-based approach to design antiviral peptides should be utilized to unearth potent anti-EV peptides.
Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). SP40 peptide was previously identified to inhibit EV-A71 strains from genotypes A, B and C. However, the stability and antiviral activity of SP40 peptide in human serum are yet to be established. To address this, we evaluated the stability and anti-EV-A71 activity of SP40 peptide after incubation in 25 % human serum. Reverse-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC/MS) were utilized to evaluate serum stability and cleavage patterns of SP40 peptide after incubation in human serum. Cell protection assay was used to evaluate the anti-EV-A71 activity of SP40 peptide after incubation in human serum and to identify the minimal active sequence of SP40 peptide that retained antiviral activity. The results showed that the SP40 peptide was stable in human serum with 56 % of the full-length SP40 peptide being detected after 48 h incubation in human serum. The SP40 peptide was mainly cleaved by exopeptidases and no endoprotease recognition sites were identified within the SP40 peptide. Cell protection assays revealed that the SP40 peptide retained substantial activity after 24 and 48 h incubation in human serum. Furthermore, the data revealed that three amino acids at the N-terminus and one amino acid at the C-terminus of the SP40 peptide were dispensable for its antiviral activity. Importantly, the four truncated peptides displayed better potency than the full-length SP40 peptide. Overall, this study provided insights into the stability and activity of SP40 peptide in human serum and will facilitate the development of SP40 peptide as an anti-EV-A71 agent.